Enzymes in the N-succinylamino acid racemase 1 (NSAR1) family catalyze the racemization of N-succinyl-D/L-amino acids, as part of a pathway for the conversion of D- to L-amino acids. The experimentally characterized enzyme from Geobacillus kaustophilus efficiently catalyzes the racemization of hydrophobic, polar, and some basic N-succinyl-D/L-amino acids. Enzymes in this family are highly similar to enzymes in the o-succinylbenzoate synthase family, and some may also function biologically as OSBSs.
Although the reaction catalyzed by this family is similar to that catalyzed by the NSAR2 family (differing only in the preference of N-succinyl arginine/lysine for NSAR2 versus N-succinyl hydrophobic amino acids for NSAR1), phylogenetic analysis suggests that these two families have independent evolutionary origins within the enolase superfamily. Not surprisingly, the amino acids responsible for substrate recognition appear to differ between the two families.
Sakai A, Xiang DF, Xu C, Song L, Yew WS, Raushel FM, Gerlt JA
Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids
▸ Abstract
Members of the mechanistically diverse enolase superfamily catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion to generate an enolate anion intermediate that is stabilized by coordination to a Mg2+ ion. The catalytic groups, ligands for an essential Mg2+ and acid/base catalysts, are located in the (beta/alpha)8-barrel domain of the bidomain proteins. The assigned physiological functions in the muconate lactonizing enzyme (MLE) subgroup (Lys acid/base catalysts at the ends of the second and sixth beta-strands in the barrel domain) are cycloisomerization (MLE), dehydration (o-succinylbenzoate synthase; OSBS), and epimerization (L-Ala-D/L-Glu epimerase). We previously studied a putatively promiscuous member of the MLE subgroup with uncertain physiological function from Amycolatopsis that was discovered based on its ability to catalyze the racemization of N-acylamino acids (N-acylamino acid racemase; NAAAR) but also catalyzes the OSBS reaction [OSBS/NAAAR; Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. In this manuscript, we report functional characterization of a homologue of this protein encoded by the genome of Geobacillus kaustophilus as well as two other proteins that are encoded by the same operon, a divergent member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes whose members catalyze the transfer an acyl group from an acyl-CoA donor to an amine acceptor, and a member of the M20 peptidase/carboxypeptidase G2 family. We determined that the member of the GNAT superfamily is succinyl-CoA:D-amino acid N-succinyltransferase, the member of the enolase superfamily is N-succinylamino acid racemase (NSAR), and the member of the M20 peptidase/carboxypeptidase G2 family is N-succinyl-L-amino acid hydrolase. We conclude that (1) these enzymes constitute a novel, irreversible pathway for the conversion of D- to L-amino acids and (2) the NSAR reaction is a new physiological function in the MLE subgroup. The NSAR is also functionally promiscuous and catalyzes an efficient OSBS reaction; intriguingly, the operon for menaquinone biosynthesis in G. kaustophilus does not encode an OSBS, raising the possibility that the NSAR is a bifunctional enzyme rather than an accidentally promiscuous enzyme.
